Chemical Composition, Antioxidant and Antibacterial Activities of Essential Oil Obtained from Chincho (Tagetes elliptica Sm) Leaves Grown in the Peruvian Andes

The chemical composition, antioxidant, and antibacterial properties of the essential oil from the leaves of Tagetes elliptica Sm., grown in Peru, were studied. The EO was extracted using steam distillation, and its chemical composition was analyzed using GC-MS, while the antioxidant activity was evaluated using the radical, scavenging capacity (DPPH and ABTS assays), and ferric reducing antioxidant power (FRAP) assays, ferrous ion chelating (FIC) activity, and the Rancimat test. The antibacterial activity against Staphylococcus aureus, Escherichia coli, and Salmonella infantis was studied using the agar well diffusion method. Twenty-seven compounds were identified in the essential oil, and the major components were cis-tagetenone (37.27%), trans-tagetenone (18.84%), dihydrotagetone (14.38%), and trans-tagetone (5.15%). With regard to antioxidant properties, the IC50 values obtained for the DPPH, ABTS, and FIC assays were 53.37, 46.38, and 22.65 mg/mL, respectively. These values were lower than those obtained for standard butylated hydroxytoluene and ascorbic acid. In the Rancimat test, antioxidant activity was achieved only at high concentration. T. elliptica essential oil showed a marked antibacterial activity against all bacterial strains at all concentrations assayed. This study demonstrated that T. elliptica essential oil could be considered as an alternative to synthetic antioxidants and antimicrobial agents in the food industry.


Introduction
The consumption of "clean label" foods formulated with natural ingredients has increased in recent years due to the increasing awareness among the population that a healthier lifestyle can improve their health, hence the demand for the food industries to innovate, look for alternatives, and use natural preservatives of plant origin to produce healthy, safe, tasty, and socially accepted foods [1,2]. Accordingly, in the food industry there is a tendency to decrease the use of synthetic additives that could be harmful to health [3]. In this sense, several studies have shown that aromatic herbs and spices, which are an important source of bioactive compounds, might be used as natural preservatives in the development of food additives or natural food ingredients due to their antioxidant and antimicrobial properties [1,4,5]. Additionally, these aromatic herbs and spices provide aroma and flavor to food and enhance the pleasure of eating.
The genus Tagetes belongs to the Asteraceae family and is represented by more than 30 species, which are adapted to live from sea level to high altitudes. It is distributed

Chromatographic Analysis
The chemical composition of T. elliptica Sm. essential oil was assessed using Hewlett-Packard 6890 gas chromatography equipment (Agilent, Santa Clara, CA, USA) equipped with a flame ionizer detector (FID) and a DB-5MS column (60 m × 250 μm, 0.25 μm film

Chromatographic Analysis
The chemical composition of T. elliptica Sm. essential oil was assessed using Hewlett-Packard 6890 gas chromatography equipment (Agilent, Santa Clara, CA, USA) equipped with a flame ionizer detector (FID) and a DB-5MS column (60 m × 250 µm, 0.25 µm film thickness). The temperature of the injector was 300 • C, whilst the temperature of detector was set at 325 • C. The oven temperature was held at 45 • C for 4 min; increased to 200 • C at 2.5 • C/min and kept for 2 min; then raised to 300 • C at 2.5 • C/min and kept for 5 min using helium as a carrier gas at 1 mL/min. Twenty µL of T. elliptica essential oil was diluted with 0.2 mL of GC-grade dichloromethane. One microliter of the diluted oil was then injected using an automatic injector into the system with a split ratio of 1:20. Quantitative data were obtained electronically from FID area data without using correction factors. All tests were performed in triplicate.
The T. elliptica essential oil was also analyzed by GC-MS with Hewlett-Packard 5975C GC-MS equipment (Agilent, Santa Clara, CA, USA). The GC-MS equipment was equipped with the same column used in the GC analysis and with the identical temperature conditions following the methodology proposed by Alves-Silva et al. [23]. The compounds were identified using the Kováts Index in reference to n-alkanes (C 8 -C 32 ), with the mass spectra of authentic standards as well as with the Wiley spectral library collection.

DPPH Radical Scavenging Method
The 2,2-Diphenyl-2-picrylhydrazyl (DPPH) scavenging activity assay was utilized, following the methodology reported by Brand-Williams et al. [24], to determine the antioxidant activity. For that, methanolic solutions of T. elliptica essential oil of six different concentrations (5,10,20,50,80 and 100 mg/mL) were assessed. Butylated hydroxytoluene and ascorbic acid (0.005, 0.01, 0.02, 0.05, 0.08 and 0.1 mg/mL) were used as standards for comparison of the antioxidant potential of the essential oil. The inhibition percentage of the DPPH radical was calculated according to Equation (1).
Additionally, inhibition (%) was plotted against the essential oil concentration in the reaction system and the concentration of essential oil required to scavenge 50% of DPPH free radicals (IC 50 value) was calculated graphically.

ABTS Radical Scavenging Method
The methodology described by Thaipong et al. [25] was used for the determination of 2,2-Aazino-bis(3-ethylbenzthiazoline-6-sulphonic acid) using a radical scavenging assay. The methanolic solutions of T. elliptica essential oil, as well as butylated hydroxytoluene and ascorbic acid, were the same as those described in Section 2.3.1. The antioxidant ability of samples was expressed as the inhibition percentage of the ABTS radical cation scavenging activity following Equation (2).
The IC 50 values were determined from a graph plotting the % inhibition against the sample concentration in the reaction system.

Ferrous Ion Chelating (FIC) Ability
The ferrous ion chelating activity of the different concentrations of (5-100 mg/mL) of T. elliptica essential oil was determined following the methodology described by Sudha et al. [26]. Ethylenediaminetetraacetic acid (EDTA) at different concentrations (0.005-0.1 mg/mL) was used as a standard for comparison of the antioxidant potential of essential oil. The ferrous ion chelating activity was calculated using Equation (3).
The IC 50 values were determined from a graph plotting the % inhibition against the sample concentration in the reaction system.

Ferric Reducing Antioxidant Power (FRAP)
The ability to reduce ferric ions of methanolic solutions of T. elliptica essential oil at six different concentrations (0.3125, 0.625, 1.25, 3.125, and 5 mg/mL) as well as butylated hydroxytoluene and ascorbic acid (0.0625, 0.125, 0.25, 0.625, and 1 mg/mL) was assessed using the methodology described by Dudonné et al. [27]. The FRAP values were expressed in terms of Trolox equivalent antioxidant capacity (TEAC) in mM Trolox/L.

Rancimat Assay
The Rancimat test was used to determine the antioxidant ability of T. elliptica Sm. essential oil, at different concentrations (5,10,20,50,80 and 100 mg/mL) against melted pork butter following the method described by Viuda-Martos et al. [28]. Butylated hydroxytoluene and ascorbic acid (0, 10, 20, 50, 80 and 100 mg/mL) were used as standards for comparison to the antioxidant potential of essential oil. The antioxidant activity index (AAI) was determined from the measured induction times, following the methodology described by Forster et al. [29] according to Equation (4).
An antioxidant activity index higher than 1 indicates inhibition of the lipid oxidation; the higher the value, the better the antioxidant activity [30].

Microbial Strains
The T. elliptica EO was individually tested against Staphylococcus aureus ATCC 25923TM, Escherichia coli ATCC 25922TM, and Salmonella infantis. The bacterial strains were cultured overnight in nutrient broth at 37 • C until a suspension of 1.0 × 10 7 CFU/mL was attained.

Agar-Well Diffusion Method
The antibacterial activity of T. elliptica EO was assessed using the agar-well diffusion method following the recommendations of Tepe et al. [31]. The bacterial inoculum (100 µL of 10 7 CFU/mL) was spread homogeneously with a sterile Digralsky loop on a Mueller Hinton agar Petri dish. A hole (6 mm in diameter) was aseptically perforated on the agar surface with a sterile tip, and 40 µL of T. elliptica EO was added into the well. After incubation for 24 h at 37 • C, all Petri dishes were observed for any zones of growth inhibition, and the diameters of these zones were measured in millimeters.

Determination of Concentration Effect
The effect of concentration (CE) was analyzed to determine which volume of T. elliptica EO showed an inhibiting effect on bacterial growth using the agar-well diffusion method. The same methodology described in Section 2.4.2 was used but adding 20, 10, and 5 mL of T. elliptica EO in each well, representing volumes 50%, 25%, and 12.5% less of the initial volume [32].

Statistical Analysis
All experiments were conducted in triplicate and the data were presented as mean ± standards deviation of triplicate determinations. The results obtained for antioxidant and antibacterial properties were examined by means of a univariate technique GLM (General Linear Model). For antioxidant and antibacterial activities, an ANOVA was applied and Tukey's post hoc test was used (p < 0.05) for the comparison between means. All determinations were analyzed using SPSS ® Statistics 22.0.0.0. software (IBM Corp., Armonk, NY, USA).

Chemical Composition of Essential Oil
The essential oil obtained from the leaves of T. elliptica Sm. had a bright light-yellow color with a pleasant odor. The yield of the essential oil recorded on a fresh weight basis was 0.23% (w/w). These values were lower than those reported for plants of the genus Tagetes cultivated in Iran (0.57 and 0.48% w/w) or in the Kingdom of Saudi Arabia (0.84% w/w) [33,34]. This variation could be due to several factors including species, part of the part used to obtain the essential oil, or environmental conditions, among others.
In the GC/MS analysis of the T. elliptica essential oil, twenty-seven compounds were identified representing 96.8% of the total essential oil ( Table 1). The main constituents were cis-tagetenone (37.27%), trans-tagetenone (18.84%), dihydrotagetone (14.38%), and trans-tagetone (5.15%). To our knowledge, there are no studies in which the chemical composition of T. elliptica essential oil has been determined. However, several studies about the chemical composition of essential oils obtained from others Tagetes species have been widely reported. In this sense, Kyarimpa et al. [35] analyzed the essential oil obtained from the aerial part of Tagetes minuta grown in Uganda. These authors reported that the main constituents of this essential oil were trans-ocimene (15.90%), I-verbenone (15%), limonene (8.02%), and tagetone (3.56%). In a similar work, Hartwig de Oliveira et al. [9] carried out a study to analyze the chemical composition of T. minuta essential oil obtained from flowers cultivated in Brazil. They found that the principal components of this essential oil were (Z)-Tagetone (70.64%), β-ocimene (11.18% w/w), and (E)-tagetone (6.24%). Omer et al. [36] investigated the chemical composition of essential oil obtained from Tagetes lucida cultivated in Egypt. These authors reported that the main constituents of this essential oil were methyl chavicol (93.18%) and linalool (2%). Gakuubi et al. [37] reported that the most abundant compounds identified in the essential oils obtained from aerial parts at the flowering stage of T. minuta cultivated in Kenya were (E)-tagetone, dihydrotagetone, and allo-ocimene. More recently, Moghaddam et al. [33] investigated the chemical composition of the essential oils obtained from leaves and flowers of Tagetes patula L. and Tagetes erecta L. grown in Iran. The main compounds found in T. patula essential oil were β-caryophyllene (24.53%), piperitenone (10.96%), and piperitone (9.66%), whilst the T. erecta essential oil was characterized by neophytadiene (17.22%), piperitone (12.52%), and β-caryophyllene (8.7%). Aati et al. [34] reported that the principal components of essential oil from T. patula cultivated in the Kingdom of Saudi Arabia were β-caryophyllene (24.1%), 2-undecanone + bornyl acetate (12.2%), and 2-nonanone (9.7%).
The scientific literature shows that the quantitative ratios of the oil components of essential oils obtained from plants of the genus Tagetes ssp. depend on several factors, including the species, crop growing location, plant development stage, harvest period, cultivation practices, parts of plant used for oil isolation, and environmental conditions, among others [38].

Antioxidant Activity of T. elliptica Essential Oil
In general terms, the antioxidant properties of essential oils could differ depending on several factors including the chemical profile and the methods assessed. In this research, five methodologies (DPPH assay, ABTS assay, FRAP assay, FIC assay, and Rancimat test), with different action mechanisms, were used. These methodologies have been widely utilized to estimate the antioxidant capacity of several products. The DPPH assay assesses the ability of phytochemicals to donate hydrogen atoms to the DPPH radical, which causes a color change in the DPPH solution. The greater the color change, the greater the antioxidant capacity, which is represented by a lower IC 50 value. Table 2 shows the antioxidant properties of T. elliptica essential oil, as well as the butylated hydroxytoluene (BHT) and ascorbic acid (AA) tested with the DPPH radical scavenging assay at different concentrations. As can be see, a concentration-dependent scavenging activity (p < 0.05) was found for T. elliptica essential oil, as well as for BHT and AA. However, it is important to highlight that the concentrations tested for the positive controls (BHT and AA) were 1000 times lower than those used for T. elliptica essential oil. This fact it is reflected in the IC 50 values (p < 0.05), which were 53.37, 0.17, and 0.02 mg/mL for T. elliptica essential oil, BHT, and AA, respectively.
The IC 50 values obtained in this work were higher than those reported by Ruiz et al. [39] for the essential oils obtained from aerial parts of T. elliptica cultivated in Peru (IC 50 = 3.4 mg/mL), as well as for the essential oils of T. minuta and Tagetes filifolia with IC 50 values of 0.8 and 20.2 mg/mL, respectively. In a similar work, Huaraca-Aparco et al. [8] reported that IC 50 values of essential oils obtained from T. elliptica and T. minuta cultivated in Peru analyzed with a DPPH assay were 2.56 and 1.77 mg/mL, respectively. Ali et al. [40] reported that essential oil obtained from the aerial parts of T. minuta cultivated in Yemen had an IC 50 value of 0.04 mg/mL. Similarly, Kyarimpa et al., [41] reported that the essential oils obtained from aerial parts of T. minuta cultivated in Uganda exhibited strong antioxidant activities when measured with a DPPH assay. With regard to the ABTS assay, significant free radical scavenging activity was observed at all concentrations of T. elliptica essential oil, and for BHT and AA, as shown in Table 2. This reduction in free radical scavenging occurs in a concentration-dependent way (p < 0.05). The IC 50 values (p < 0.05) calculated with this methodology were 46.38, 0.016, and 0.017 mg/mL for T. elliptica essential oil, BHT, and AA, respectively. It is important to highlight that the IC 50 values achieved with the ABTS assay were less than those obtained with the DPPH assay. These variations between the two methodologies used may be explained by the fact that they have different action mechanisms. As mentioned by Platzer et al. [42], in the ABTS assay the reaction involves e − transfer and occurs at a much faster speed compared with DPPH radicals, whose reaction could be attributed to the hydrogen donating capacity of several compounds present in T. elliptica essential oil. As occurs in the DPPH assay, the IC 50 values obtained in the ABTS assay for T. ellipitica essential oil were higher than those reported by Huaraca-Aparco et al. [8], who reported that the IC 50 values for T. elliptica and T. minuta essential oils were 41.06 and 21.02 mg/mL. On the other hand, Hartwig de Oliveira et al. [9] reported that the IC 50 value attained in the ABTS assay for T. minuta essential oil obtained from flowers cultivated in Brazil was 0.1 mg/mL. Other mechanism of antioxidant action is the chelation of transition metals. These metals may promote the lipid peroxidation via the generation of initiator species and increasing peroxidation. Thus, the study of the metal ion-chelating properties ( Table 2) showed that T. elliptica essential oil was capable of chelating Fe +2 when high concentrations (12.5-25 mg/mL) were assayed, with statistical differences (p < 0.05) between all samples. At low concentrations (1.25-5 mg/mL), no chelating effect was observed. Similarly, at low concentrations (0.005-0.02 mg/mL) of EDTA used as standard, no chelating activity was observed. The IC 50 values for T. elliptica essential oil and positive control EDTA were 22.65 and 0.06 mg/mL, respectively.
The ferric reducing properties of T. elliptica essential oil, BHT, and AA were attained utilizing the FRAP methodology as shown in Table 3. As can be seen, the ferric reducing capacity for all samples assayed occurred in a concentration-dependent manner. These results agree with those reported by Hartwig de Oliveira [9], who reported that the essential oil obtained from flowers of T. minuta showed a reducing potential in a concentrationdependent manner possibly due to an electron transfer mechanism. Mlala et al., [43] reported that the essential oil obtained from leaves of T. minuta cultivated in South Africa had a ferric reducing capacity of 63.56 µg ascorbic acid equivalent/mL of extract. Rancimat assay is a cheap and simple methodology that needs low sample volumes and attains reproducible results. Table 4 shows the values obtained for the antioxidant activity index (AAI) of animal fat with T. elliptica essential oil added, and with the standard BHT and AA essential oils added. Thus, the longer the induction period of the animal butter added with the essential oil, BHT, or AA standards compared to the control, which is pure butter, the more potent the antioxidant capacity of that compound will be. The AAI decreased in the order AA > BHT > T. elliptica Sm. According to this methodology, only concentrations of 5 and 10 mg/L of T. elliptica Sm. essential oil displayed antioxidant activity, with AAI values of 1.01 for both concentrations. These values were lower than those obtained for standard compounds (Table 4). It is important to highlight that T. elliptica Sm. essential oil showed pro-oxidant effects at concentrations ranging between 20 and 100 mg/mL). The pro-oxidant effect of essential oils is widely described in the scientific literature, as described in the works of Poma et al. [44] and Kong et al. [45]. The results obtained show that the essential oils obtained from aerial parts of T. ellipitica had a moderated antioxidant activity. These antioxidant properties could be attributed to a high content of acyclic monoterpenes ketones, including cisand transtagetenone and tagetone, found in their composition as well as the synergistic action among several major and minor components [21,46]. However, the mechanism of action by which the compounds present in essential oils exert their antioxidant effect is not yet clearly understood. In this way, several mechanisms have been proposed, principally their redox properties, that play a significant role in absorbing and neutralizing free radicals, quenching singlet oxygen, and decomposing peroxides, as reported Jugreet et al. [47].

Antibacterial Activity of T. elliptica Essential Oil
The agar-well diffusion method was used to analyze the antibacterial activity of T. elliptica essential oil against Gram-positive and Gram-negative bacteria as shown in Table 5. These bacterial strains, S. aureus and E. coli, are considered model pathogenic bacteria for the evaluation of antimicrobial activity of plant extracts [48] while S. infantis is a pathogenic bacterium responsible for zoonoses transmissible to humans, with high resistance to antibiotics. Table 5. Antibacterial activity of T. elliptica essential oil using the agar-well diffusion method.

Diameter (Mean and SD) of Inhibition Zone (mm) Including
Well T. elliptica EO showed antibacterial activity against all strains studied. Thus, Staphylococcus aureus (Gram-positive) had the highest inhibition halo (p < 0.05), while E. coli and S. infantis showed lower inhibition diameters with no difference (p > 0.05) between them. To the best of our knowledge, this is the first work to determine the antibacterial properties of the essential oil obtained from leave of T. elliptica. Nevertheless, as occurs with the chemical composition, the antibacterial properties of several other species of the genus Tagetes have been widely studied. Thus, Shirazi et al. [49] assessed the antibacterial activity of essential oils obtained from the leaves and flowers of Tagetes minuta cultivated in Iran. These authors reported that the minimal inhibitory concentration against Salmonella typhi, Escherichia coli, Staphylococcus aureus, and Bacillus subtilis were 150, 165, 67, and 75 µg/mL of T. minuta EO, respectively. Similarly, Gakuubi et al. [37] reported that the essential oil of T. minuta obtained from the leaves, flowers, and stem (at the flowering stage), with a volume added to the disk of 10 µL, had a very important antibacterial activity against Pseudomonas savastanoi, Xanthomonas axonopodis, and Xanthomonas axonopodis with inhibition halos of 41.83, 26.83, and 26.83 mm, respectively. More recently, Safar et al. [50] reported antibacterial activity of Tagetes patula EOs against several bacterial strains, including Serratia fonticola, Klebsiella pneumoniae, Proteus mirabilis, Escherichia coli, and Staphylococcus aureus, with minimum inhibitory concentration values ranging between 0.16 and 0.64 µL/mL.
The results obtained in this work showed that T. elliptica essential oil was more effective against Gram-positive bacteria when compared to Gram-negative. In this sense, several studies on the antibacterial properties of essential oils obtained from plants of the genus Tagetes, as well as other plant species, have commonly reported that the Gram-positive bacteria were more susceptible to the effects of EOs in comparison to Gram-negative bacteria [49,51,52] due to the fact that the Gram-negative bacteria have an outer membrane that restricts the diffusion of hydrophobic substances [53]. In view of the huge number of diverse groups of chemical compounds found in T. elliptica essential oil, it is most likely that their antibacterial activity is not attributable to one specific compound. In addition, the mechanism of action of essential oils obtained from plants of the genus Tagetes is still unknown. Several studies have reported that the cell membrane is the principal structure of the microorganism which is damage by the most of chemical compounds found in essential oils [54]. In addition, several chemical compounds found in essential oils may change the successive process of the synthesis of macromolecules, including proteins, polysaccharides, DNA, or RNA, which finally provoke the cell's death [55]. Therefore, Senatore et al. [56] reported that that terpenoids such as dihydrotagetones, tagetones, and ocimenones, which were found in T. elliptica essential oils, are sufficient to account for the observed antibacterial activities of this essential oil due to the fact that they may provoke several impairments to the cell membrane, including some distorted projections on the cell membrane, and the cell morphology.
The concentration effect of the essential oil from T. elliptica on the three bacterial strains studied is shown in Table 5. All the concentrations assayed produced inhibition halos against all bacterial strains. In the case of S. aureus, this fact occurs in a concentrationdependent manner (p < 0.05). However, in the case of E. coli and S. infantis, no statistical differences were found between any of the concentrations analyzed.
As mentioned in Ruiz-Navajas et al. [32] it is important to notice that, when the essential oils are utilized as antimicrobial agents, their effectiveness is reduced when these phytochemicals are incorporated into complex matrices such as foods, without forgetting the impact on the organoleptic properties may have. In addition, several questions about toxicity and safety should be taken into consideration.

Conclusions
The results obtained in this work demonstrated that T. elliptica Sm. essential oil is a good source of important bioactive compounds (cis-tagetenone, trans-tagetenone, and dihydrotagetone) and possesses a moderate antioxidant capacity with different possible action mechanisms. In the same way, this essential oil has strong antibacterial properties against both Gram-negative and Gram-positive bacteria. However, further studies would be necessary to clarify the compound or compounds responsible for both antioxidant and antibacterial activity.
In any case, in view of these properties, this essential oil could be considered a safe and eco-friendly alternative to synthetic antioxidants and antimicrobial agents in the food industry. Despite the promising results obtained in in vitro assays, more detailed studies of the mechanisms of action of T. elliptica essential oils would be beneficial to achieve their potential as a natural preservative in the development of food products. This is because, when the essential oils are added to more complex matrices such as food, they tend to reduce both antioxidant and antibacterial properties due to possible interactions between food and essential oil components. This would make it necessary to add higher concentrations to achieve the same effect, which could have undesirable effects on the organoleptic characteristics of the product.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.